Method for maintenance and propagation of germline stem cells using members of the TGF-beta family of growth factors

ABSTRACT

The TGF-β family of growth factors, particularly the bone morphogenetic protein (BMP)-2/4 homolog decapentaplegic (dpp), are specifically required to maintain germline stem cells and promote their division. Overexpression of dpp blocks germline stem cell differentiation. Mutations in dpp or its receptor saxophone accelerate stem cell loss and retard stem cell division. dpp signaling is directly received by germline stem cells, and thus dpp signaling helps define a niche that controls germline stem cell proliferation.

RELATED APPLICATIONS

[0001] This application claims priority from provisional U.S. Appln. No.60/094,008, filed Jul. 24, 1998.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to members of the transforming growthfactor-β family and their regulation of cell division, cell survival,and the specification of cell fates. Particularly, the invention relatesto the bone morphogenetic protein (BMP)-2/4 homolog deca-pentaplegic(dpp) and its role in the maintenance of stem cells. For example, adpp-based method for maintenance and controlling the division ofgermline stem cells, and a dpp-based method for defining a niche thatcontrols germline stem cell proliferation are disclosed. Additionally,the invention provides a model of ovarian tumor development. Theinvention further relates to a dpp-based method for propagating stemcells in an undifferentiated state in vivo or by culturing in vitro.

[0004] 2. Description of Related Art

[0005] In many adult tissues that undergo continuous cell turnover, apopulation of stem cells is responsible for replacing lost cells.Because of their pivotal role in controlling growth and neoplasia, themechanisms regulating stem cell function are of great interest (reviewedby Potter and Loeffler, 1990; Doe and Spana, 1995; Lin, 1997; Morrisonet al., 1997). Two mechanisms have been proposed to maintain stem celldivisions and regulate the differentiation of stem cell daughters:intrinsic factors and extracellular signals. Asymmetrically localizedintrinsic factors help specify the fates of neuroblast daughters inDrosophila embryos (Doe and Spana, 1995). Extracellular signals fromsurrounding cells mediated by cell surface-associated ligands anddiffusible factors are frequently involved (Potter and Loeffler, 1990;Morrison et al., 1997). The identification of several of these factorshas made it possible to culture some types of stem cell in vitro.

[0006] The Drosophila ovary presents an excellent system for studyingtwo distinct groups of stem cells that remain active during much ofadult life (reviewed by Spradling et al., 1997). The adult ovarycontains 14-16 ovaribles each with a germarium at the tip, within whichthe germline and somatic stem cells are located. Two or three germlinestem cells, located at the anterior tip of the germarium, divideasymmetrically to generate all germline cells in the ovariole (Wieschausand Szabad, 1979; reviewed by Lin, 1997). Stem cell daughters known ascystoblasts undergo four rounds of synchronous division to producegroups of two, four, eight, and eventually 16 interconnected cystocytes,the precursors of ovarian follicles (reviewed by de Cuevas et al.,1997). Two somatic stem cells residing in the middle of the germariumgive rise to all the somatic follicle cells (Margolis and Spradling,1995); their equivalent in the testis are cyst progenitor cells. Threetypes of mitotically quiescent somatic cells are located in the vicinityof the stem cells: terminal filament and cap cells contact the germlinestem cells, while inner sheath cells lie more posteriorly and contactboth stem cell types.

[0007] Germline stem cell division is known to involve intrinsicmechanisms. This division and subsequent cystocyte divisions arephysically unequal due to the segregation of fusomes rich in membraneskeleton proteins such as α-spectrin and an adducin homolog encoded byhu-li tai shao (hts) (reviewed by de Cuevas et al., 1997). The roundfusome (or “spectrosome”) characteristic of stem cells changes shape ascyst development proceeds, allowing cysts at different stages to beidentified. The bag of marbles (bam) gene is highly expressed only inthe stem cell daughter (McKearin and Spradling, 1990). The loss of Bamprotein in cystoblasts prevents their differentiation, causing germlinetumors to form (a “tumor” in Drosophila is a large clump ofproliferating cells, the term does not imply these cells are cancerous).The genes pumilio (pum) and nanos (nos), encoding translationalregulators, also play critical roles in the formation and maintenance ofgermline stem cells (Lin and Spradling, 1997; Forbes and Lehmann, 1998).

[0008] Less is known about the intercellular signals that control stemcell proliferation. Two important signaling molecules, Hedgehog (Hh) andWingless (Wg) (reviewed by Perrimon, 1995; Cadigan and Nusse, 1997), areexpressed in terminal filament and cap cells (Forbes et al., 1996a and1996b). Hh signaling is critical for proliferation and differentiationof follicle cells, but it remained to be determined at the time thepresent invention was made whether somatic stem cells or their daughtersare regulated (Forbes et al., 1996a and 1996b). The role of thesesignals in the germ line was even less clear because ectopic expressionof hh did not appear to interfere with the function of germline stemcells (Forbes et al., 1996a).

[0009] Members of the transforming growth factor-β (TGF-β) family,including TGF-βs, activins, and the bone morphogenetic proteins (BMPs),elicit a broad range of cellular responses including the regulation ofcell division, survival, and specification of cell fates (reviewed byMassague et al., 1996; Hogan, 1996a). TGF-βs were previously identifiedas repressing the proliferation of stem cells as assayed by either invitro cultures or in vivo ectopic expression (Potter and Leoffler, 1990;Morrison et al., 1997). Inactivation of BMP-4 and its receptor BMPR inmice resulted in embryonic lethality for homozygous mutants. (Winnier etal., 1995; Mishina et al., 1995), but no effect on stem cells was noted.

[0010] Similarly dpp, encoding a vertebrate BMP-2/4 homolog inDrosophila, functions as a local signal as well as a long-distancemorphogen to pattern the early embryo and adult appendages by regulatingcell proliferation and cell fate determination (Padgett et al., 1987;reviewed by Lawrence and Struhl, 1996). dpp is expressed in an anteriorsubset of follicle cells, and is required for establishing egg shape andpolarity during late stages of oogenesis (Twombly et al., 1996). But aneffect of dpp on maintaining and propagating stem cells, instead ofcausing their differentiation, has not been previously shown.

[0011] Major participants in the dpp signaling pathway have beenidentified: saxophone (sax) and thick veins (tkv) encode type Iserine/threonine kinase transmembrane receptors, whereas punt encodes atype II serine/threonine kinase transmembrane receptor (Brummel et al.,1994; Nellen et al., 1994; Penton et al., 1994; Xie et al., 1994;Ruberte et al., 1995; Letsou et al., 1995). mothers against dpp (mad),Medea (Med), and Daughters against dpp (Dad) encode a family ofconserved TGF-β transducers (Sekelsky et al., 1995; Tsuneizumi et al.,1997; Hudson et al., 1998; Wisotzkey et al., 1998; Das et al., 1998;Inoue et al., 1998), collectively known as Smads. Smads are proteinswhich transduce signals on behalf of TGF-β family members, or inhibitTGF-β signal transduction. A paradigm for TGF-β signal transduction hasbeen developed from several experimental systems (Heldin et al., 1997).In Drosophila, Dpp binds both type I and II receptors to allow theconstitutively active Punt kinase to phosphorylate and activate type Ikinases, which phosphorylate Mad. The phosphorylated Mad brings Med intothe nucleus as a transcriptional activator to stimulate dpp target geneexpression.

[0012] Enhancing Dpp or other BMP-like signaling activities can beachieved by reducing the presence of Dad-like proteins, such as humanSmad6 and Smad7. Vertebrate Smad6 and Smad7 interact with type Ireceptors, and are known to inhibit both TGF-β and BMP signaling incultured cells and frog embryos. Thus, disinhibition of TGF-β familymembers by inhibiting certain Smads promotes BMP-like signalingcascades. Additionally, Dpp or other BMP-like signaling activities maybe increased by enhancing the function of Dpp or BMP receptors, such asSax, Tkv, and Punt in Drosophila, and BMP receptors BMPR-II, ActR-II,Act-IIB, BMPR-IA, and ActR-I in humans. Other downstream positiveregulators of Dpp or BMP signaling include Mad, Med, Dad, and Schnurriproteins in Drosophila, and Smad1, Smad4 and Smad5 in humans. See reviewby Padgett (1999).

[0013] Therefore, to address the prior art's failure to identify andcharacterize factors involved in germline stem cell maintenance andpropagation, we now disclose that a member of the TGF-β family of growthfactors and its signaling pathway unexpectedly provide this essentialfunction.

SUMMARY OF THE INVENTION

[0014] It is an object of the invention to maintain and/or propagatestem cells by stimulating signaling through a bone morphogenetic protein(BMP) signaling pathway. In this manner a population of stem cells canbe maintained in vivo or in vitro, and/or expanded.

[0015] Methods for maintaining germline and somatic stem cells of anorganism are provided by stimulating a bone morphogenetic protein (BMP)signaling pathway.

[0016] The signal transduction pathway associated with a BMPspecifically binding to a receptor may involve phosphorylation ofserine/threonine residues (e.g., kinases, phosphatases) and a cascade ofcomponents of the pathway (i.e., signal transducers such as, forexample, transcription factors) which communicate that signal. Forexample, a signal may be communicated from BMP binding at the cellsurface to the nucleus where gene expression of downstream targets areeither activated or inhibited. Thus, BMP signaling may be modulated atone or more steps in this pathway, or by affecting upstream regulatorsor downstream targets of this signaling pathway. Modulation (i.e.,stimulation or repression) of BMP signaling may be accomplished directlyon the stem cell or indirectly through other cells in a mixed cellpopulation (e.g., feeder layer).

[0017] Properties of the stem cell may be maintained by stimulating BMPsignaling. Furthermore, stem cells may be increased in abundance and/orincreased in lifetime by such stimulation. Conversely, stem cells ortumor cells in a population may be reduced in total number orconcentration, or even eliminated at the limit of detection, byrepressing BMP signaling.

[0018] Stem cells may also be propagated and isolated according to theinvention.

[0019] Our invention addresses the problem of restricted access to andlimited numbers of stem cells. The ability to maintain and to propagatestem cells facilitates genetic manipulation and the characterization ofthese rare cells.

DESCRIPTION OF SPECIFIC EMBODIMENTS

[0020] The present invention provides a method for maintaining andcontrolling the division of germline stem cells in which dpp can providean essential role. Further, it provides a model of ovarian tumorformation in which overexpression of dpp produces ovarian stem celltumors. Clonal analysis demonstrates that downstream components (i.e.,signal transducers) of the dpp signaling pathway are requiredcell-autonomously in the germline stem cells for their division andmaintenance. This invention also provides a method for control of acellular niche by BMP signaling, in which germline stem cells areregulated by, for example, a dpp signal that likely derives fromsurrounding somatic cells.

[0021] Stem cells are thought to be regulated by positive and negativediffusible factors, but the functions of most of these factors havenever been demonstrated in vivo. The present invention provides a methodin which Dpp directly signals to maintain Drosophila germline stem cellsand stimulate their division. The experiments of the examples were madepossible by a clonal cell marking method that allows the function ofstem cells and their progeny to be examined directly over many cellgenerations. In addition to the dpp signal, known components in the dppsignal transduction pathway were shown to be required in these adultstem cells. This action appears to be specific to stem cells, since germcells lacking dpp pathway components were still able to form 16-cellcysts. The examples demonstrate that a TGF-β-like molecule functions asa stem cell growth factor.

[0022] dpp signal transduction is required for maintaining stem cells,on which Dpp may act in several distinct ways. Signaling preventsgermline stem cells from differentiating into cystoblasts and gametes.The examples show that overexpressed dpp prevents stem celldifferentiation, while reduction of dpp function promotes stem celldifferentiation. An attractive candidate target of the dpp signaltransduction pathway is the Bam protein, which is normally synthesizedat much higher levels in cystoblasts than in stem cells (McKearin andOhlstein, 1995). The forced expression of Bam in germline stem cellscauses them to differentiate in a manner very similar to that caused byreductions in dpp signaling (Ohlstein and McKearin, 1997). Thus, dppsignaling may negatively regulate Bam protein levels in germline stemcells. Two other genes, pum and nos, are required to form and maintaingermline stem cells (Lin and Spradling, 1997; Forbes and Lehmann, 1998).In the embryo, both proteins work together to repress the translation oftarget genes such as hunchback (hb) (Baker et al., 1992; Murata andWharton, 1995). In the ovary, dpp signaling may downregulate Bam througheffects on the Nos/Pum pathway or by an independent mechanism. However,genes throughout the dpp pathway are required, including two nucleartranscription factors, suggesting that the action of the pathway is ontranscription of target genes. Also see reviews by Attisano and Wrana(1998), Kawabata et al. (1998), and Padgett et al. (1998).

[0023] dpp may also function to maintain a specialized associationbetween the stem cells and basal terminal filament cells. Such anassociation has been postulated to hold the stem cells at the anteriorof the germarium, while daughter germline cells all move posteriorly andeventually leave the germarium. The results presented herein indicatethat the stem cell loss is due to differentiation. Possibly, dppsignaling via its receptor regulates the expression of adhesionmolecules that reside on the cell surface or of cytoplasmic proteinsthat indirectly promote stem cell adhesion.

[0024] dpp signaling also may act to stimulate stem cell division. dppsignaling stimulates cell proliferation at several points duringDrosophila development. In the wing imaginal disc, it is essential forcell proliferation and/or survival (Burke and Baster, 1996), whereas itpromotes the G2-M transition in the morphogenetic furrow of thedeveloping eye disc (Penton et al., 1997). Consistent with such arequirement, mad mutants have greatly reduced imaginal discs, shortenedgastric caeca, and small brains (Sekelsky et al., 1995). The requirementfor dpp signaling disclosed herein suggests that adult stem cells usestrategies similar to those of embryonic and larval somatic cells toregulate proliferation. For example, dpp stimulates the rate of celldivision for stem cells.

[0025] During aging, the number and activity of stem cells are thoughtto be reduced. The examples indicate that the level of dpp signalingcontrols the life span and division rate of germline stem cells. Reduceddpp signaling caused premature stem cell loss. Perhaps more surprisingis the observation that putative increases in signaling, caused byremoval of Dad protein activity from stem cells, permitted stem cells tobe maintained longer. This finding suggests that dpp signaling not onlyis necessary, but may sometimes be rate limiting for stem cellmaintenance. The illustrative examples demonstrate for the first time amethod in which stem cell life span has been extended in an intactorganism.

[0026] These results suggest that it may be possible to extend the lifespan of stem cells, a process that could be of therapeutic significance.For example, drugs that upregulate BMP signaling to stem cells mayenhance fertility in humans and animals, such as male fertility inpatients with reduced numbers of germline stem cells (basal cells). Suchdrugs may ameliorate hematologic conditions caused by reduced stem cellfunctioning, for example aplastic anemias, agammaglobulinemia, andrelated conditions. Drugs enhancing BMP signaling may enhance woundhealing. Aging-related pathologies caused by loss of stem cells, such ashair loss, loss of muscle mass, reduction of blood cell numbers, and theaging of the skin and other stem cell-dependent tissues could be treatedby increasing BMP signal transduction. Compounds enhancing BMP signalingmay increase the average lifespan of an organism.

[0027] One method for the enhancement of dpp signal transduction may befacilitated by removal of the dpp inhibitor Dad or other Dad-likeinhibitory protein activity (inhibitory Smad activity) from the germlinestem cells. Dad is induced by dpp signaling, but then acts todowregulate the very pathway that activated its production. This methodcould also be practiced with other negative regulators of the dppsignaling pathway and, in particular, inhibitory Smads. In contrast,brinker (brk) is a target gene repressed by dpp signaling and, becauseit is itself a transcriptional repressor, the net effect of repressingexpression of the Brk repressor is to upregulate Brk-regulated targetgenes (Minami et al., 1999; Campbell et al., 1999; Jazwinska et al.,1999). This results in the increased production of Brk-regulated targetgenes following dpp signaling. Hence, BMP signaling can be stimulated orrepressed by appropriate manipulation of Smads or target genes which areregulated by BMP signaling (i.e., increasing or decreasing their effectsas appropriate to achieve stimulation or repression of BMP signaling).The roles of Dad and Brk, like the rest of the pathway, appear to beconserved in mammals.

[0028] Drugs that inhibit BMP signaling to stem cells may be usefulchemotherapeutic agents. For example, drugs inhibiting BMP signalingpathways may be useful therapies against teratocarcinoma by causing stemcell differentiation. As another example, drugs which inhibit BMPsignaling may be successful treatments against ovarian germline tumorsdependent upon BMP signaling for continued growth.

[0029] Increased or decreased BMP signaling to stem cells might allowpopulations of stem cells to expand prior to bone marrow transplant,thereby increasing the chances of successful transplantation andreducing the amount of donor marrow required. Further, control of BMPsignaling pathways may permit stem cells other than those in bone marrowto be removed from a patient, expanded in vitro, and subsequentlyreintroduced in to the patient to repair tissues damaged by injury ordisease, such as Parkinson's disease.

[0030] Bone marrow from patients with hematologic tumors, such aslymphoma and leukemia, could be tested for BMP sensitivity. Positivetest results for BMP sensitivity would allow steps to be taken to avoidpotential side effects of anti-BMP treatment in vivo. For example,marrow removed from the patient could be cleansed of tumors cells byinhibiting BMP signaling, thereby inducing differentiation of tumorcells and reducing the tumor burden. The cleansed marrow wouldsubsequently be returned to the patient in an autologous bone marrowtransplant. Such differentiation therapy could also be used for solidtumors like sarcoma, carcinoma, and neuroglioma to reduce tumor burden.Therapy may be use alone or in association with other treatments suchas, for example, chemotherapy, hyperthermia, or radiation whichpreferentially kills rapidly dividing cells and surgical resection oftumor.

[0031] Upregulation of BMP signaling to stem cells may permit the growthof germline stem cells in culture, useful in, for example, generatingtransgenic animals. Such techniques are especially useful in organismswhich have not traditionally been used as genetic models of developmentand disease.

[0032] The ability to expand stem cell niches by overexpression of TGF-βmembers, such as dpp may allow rare human stem cells, or alternativelyrare stem cells of any species, to be purified and propagated followingtransfer into living Drosophila, which have been genetically engineeredto serve as hosts.

[0033] Beside biomedical research and treatment, other uses for thepresent invention include agriculture and wildlife conservation. Stemcells could be provided in or obtained from humans, primates (e.g.,bonobo, chimpanzee, gorilla, macaque, orangutan), companion animals(e.g., dog, cat), and farm/laboratory animals (e.g., cattle, donkey,goat, horse, pig, sheep; amphibians such as frog, salamander, toad;birds such as chicken, duck, turkey, fishes such as carp, catfish,medaka, salmon, tilapia, tuna, zebrafish; lagomorphs such as hares,rabbits; rodents such as mice, rats).

[0034] Stem cells could be maintained and/or maintained in anappropriate niche or in culture, and used as a source of nuclei forcloning progeny organisms via nuclear transfer or a source of cells forpropagation of mosaic organisms via embryo aggregation. Thus, Dpp orrelated BMPs provide a means for growing stem cells in vitro or in vivofor cloning animals.

[0035] BMP signaling is unlikely to be confined to one type of BMP andonly type of BMP receptor because of the ability of evolutionarilydiverged components of the BMP signal transduction pathway or differenttypes of BMPs, BMP receptors, and SMADs to be functional equivalents ofeach other. For example, there appears to be crosstalk between Dpp/Tkvsignaling and Gbb/Sax signaling (Haerry et al., 1998) and one signaltransducer acts in different signaling pathways (Lagna et al., 1996).For example, a mixture of BMPs could be added to defined culture mediumor be present in conditioned culture medium such that Dpp and Gbb wouldsynergize in initiating BMP signaling through more than one differenttypes of BMP receptor. As another example, one type of signal transducercould stimulate signaling through more than one different types of BMPreceptor.

[0036] To stimulate BMP signaling, a positive signal transducer could beincreased in expression (e.g., more transcripts and/or translatedproducts) or mutated to a gain-of-function phenotype to increaseactivity of that signal transducer, while a negative signal transducercould be decreased in expression (e.g., fewer transcripts and/ortranslated products) or mutated to a loss-of-function phenotype todecrease activity of that signal transducer. Alternatively, a downstreamtarget gene of BMP signaling could be directly activated or inhibitedwithout BMP binding to its receptor by genetic engineering using atransactivator like GAL4 binding its UAS or ecdysone receptor bindingupstream of the target gene. Similar techniques in mice involveinduction with tetracycline or FK506.

[0037] Another method would be to increase endogenous BMP activity inthe cells or to increase exogenous BMP activity outside the cells,especially if ligand is the limiting component in BMP signaling. Forexample, BMP expression may be increased in a stem cell and stimulateBMP signaling through an autocrine mechanism. Alternatively, BMPexpression may be increased in a non-stem cell or a feeder cell, andthen BMP activity could be secreted and taken up by the stem cell orbrought into contact with the surface of the stem cell. BMP could alsobe added to the extracellular space or culture medium. BMP activity maybe increased to stimulate BMP signaling by at least about 10%, 50%,100%, or 200% as compared to the amount normally present in the animalor the culture.

[0038] Properties of the stem cell which may be maintained include thefollowing: pluripotency, totipotency, committing to one or moredifferentiating cell lineages, giving rise to multiple different typesof progenitors and/or differentiated cells, contributing to thegermline, and combinations thereof. Thus, the growth and/or survival ofstem cells may be maintained without commitment to a program ofdifferentiation, while retaining the capacity to differentiate normallyunder appropriate conditions following reduction or elimination of BMPsignaling. More simply, stem cells in a population may be expanded intotal number or concentration relative to non-stem cells (i.e., anincrease in abundance), extended in the time between a stem cell's birthand its death or apoptosis (i.e., an increase in lifetime), orcombinations thereof. Conversely, stem cells or tumor cells in apopulation may be reduced in total number or concentration, or eveneliminated at the limit of detection, by repressing BMP signaling.

[0039] Stem cells made according to the present invention may betotipotent or pluripotent, male or female, germline or somatic, dividingor quiescent, vertebrate or invertebrate, present in situ or isolated,partially or substantially purified of differentiate cells, andcombinations thereof. Proliferating stem cells are diploid, enteringmeiosis and the later stages of gametogenesis is part of the program ofdifferentiation for male or female germline stem cells that is preventedby the present invention. Stem cells may be present in or obtained fromtestis, ovary, especially apical tips of Drosophila testes and/orovarioles, or other adult or embryonic tissues. By differentiating, stemcells may differentiate into cells of the hematopoietic, immune, ornervous systems or the like. Preferably, stem cells maintained and/orpropagated by the present invention retain the potential to laterdifferentiate and thereby contribute to oogenesis or spermatogenesis,all three germ layers (i.e., endoderm, mesoderm, ectoderm), multipledifferentiated cell lineages, and combinations thereof.

[0040] Somatic cells include terminal filament cells, cap cells, andinner sheath cells from the ovary and hub cells from the testis.Preferably, the present invention reduces the proportion of somaticcells in a population relative to germline cells during maintenanceand/or propagation. A niche defined by surrounding somatic cells or afeeder layer comprised of somatic cells may provide cell contact andother extracellular signals to maintain and/or propagate germline cells.A feeder layer may be provided that provides certain essentialextracellular signals by, for example, genetically manipulating culturedcells to express and secrete a BMP which then binds to its receptor onthe stem cells.

[0041] Cell populations may be derived from the germline or somatic (ormixed), male or female, dividing or quiescent, vertebrate orinvertebrate, present in situ or isolated, partially or substantiallypurified, and combinations thereof. Preferably, cell populations includecells expressing one or more BMPs; more preferably, BMP is secreted bynon-stem cells and binds to receptors of stem cells to stimulate BMPsignaling. Thus, stem cells of the present invention contain receptorsfor BMP, especially Dpp or a homolog, or are at least responsive to BMPsignaling.

[0042] Besides mammals, amphibians, birds, and fishes, other organismsmay be used in the present invention such as invertebrates like worms(e.g., Helminthes, Nematodes) and insects (e.g., Anopheles, Drosophila).In particular, comparison of components of the BMP signaling pathway,upstream regulators, and downstream targets show them to be highlyconserved (Bitgood and McMahon, 1995; Padgett et al., 1998). Thus, thepresent invention should not be limited in its usefulness to Drosophilamelanogaster. Other species which show conservation of dpp (Newfeld etal., 1997) and are likely to be useful are D. simulans, D.pseudoobscura, and D. virilis. For metazoan species in which there hasbeen a diligent search, a dpp-like gene has been identified.

[0043] Mammalian homologs of dpp, glass bottom boat (gbb), and screw(scw) have been identified as BMP-2/4, BMP-5/8, and BMP-6, respectively(Hoffmann, 1997; Raftery and Sutherland, 1999; Wharton et al., 1999). Amammalian serine/threonine kinase receptor has been identified thatspecifically binds BMP-2 and BMP-4 (Yamaji et al., 1994). Other relatedmembers of the TGF-β family, their receptors, or other components oftheir signaling pathways, might also be used in the present invention.See also U.S. Pat. Nos. 5,011,691, 5,013,649, 5166,058, 5,168,050,5,216,126, 5,324,819, 5,354,557, 5,635,372, 5,639,638, 5,650,276, and5,854,207.

[0044] Furthermore, mutational analysis and determination ofstructure-function relationships have identified conserved residues andessential residues for Dpp signaling (Wharton et al., 1996). Bacteriallyexpressed Dpp can be refolded, then biochemically and biophysicallycharacterized (Groppe et al., 1998). Homologs of a member of the BMPfamily, their receptors, and other components of the signaling pathwaycan be identified by a high level of structural conservation when aminoacid sequences are compared, and/or functional conservation whenhomologs rescue mutant phenotypes or otherwise replace BMP activity.

[0045] Cell types have been identified by markers and are wellcharacterized by genetic mutants and developmental studies. Stem cellsmay be provided in situ as part of an intact organism or they may becultured in vitro. Germline stem cells and surrounding cells may be froman adult (e.g., ovary, testis) or an embryo. For in vitro culturing,cells may be obtained directly from an organism (i.e., primary culture)but it would be convenient to passage them through several cultures(e.g., at least five, ten, or twenty times) to expand their number(e.g., at least two, ten, or 100 times more than the original number).

[0046] Stem cells may be isolated from a donor organism with or withoutincreasing cell number by stimulating BMP signaling; manipulated duringtransient in vitro culturing under conditions for maintenance and/orpropagation by treating with one or more chemicals, introducing geneticmaterial, fusing with another cell, mutating one or more genes,selecting a desired genotype or phenotype, or combinations thereof; andtransplanting stem cells back into a host which is identical to thedonor (i.e., autologous transplantation), similar to the donor butdifferent (i.e., allogeneic transplantation), or is totally differentfrom the donor (i.e., xenogeneic transplantation). In vitro cultureconditions, genetic engineering of Drosophila by transfection andsite-specific recombination, and cell or nuclear transplantation areknown in the art.

[0047] For Drosphila, there are only about 10 germline stem cells pertestis and about 32-48 germline stem cells per ovary (i.e., there areabout 16 ovarioles per ovary and about two or three germline stem cellsper ovary). The present invention provides greatly increased numbers ofstem cells to be produced in vivo in an adult or embryo, and thencultured in vitro. In vitro culture of cells may be carried out byinitially generating flies with a large number of germline stem cells ineach ovariole. Then ovaries may be removed surgically into sterileculture medium and the germ cells released (they do not adhere and,thus, do not need to be disaggregated). Alternatively, disaggregatedembryos may also be used as a source of germline stem cells. Althoughthe number of germ cells per embryo is similar to the number per ovaryand testis, it is possible to start with 100,000 embryos but only a fewhundred gonads can be easily obtained. Schneider (1972) shows derivationof a cell line from Drosophila.

[0048] Drosophila cells may be plated into small wells containing feederlayers of cells expressing Dpp (e.g., Panganiban et al., 1990) or Hh(e.g., Lee et al., 1994), or culture media prepared by conditioning themedia with cells secreting soluble factors or simply adding arecombinantly produced soluble factor (e.g., Dpp produced according toGroppe et al., 1998). In vitro culture media for growing Drosophilacells are commercially available such as, for example, Schneider'sDrosophila medium. Drosophila cells can also be adapted and grown inmammalian tissue culture media (Spradling et al., 1975; Lengyel et al.,1975). Drosophila cells can be transfected like mammalian cells (Burkeet al., 1984). Constructs and strategies for homologous recombination insomatic, embryonic stem (ES), and embryonic (EG) cells could be adaptedfor use with in vitro cultured Drosophila cells (Capecchi, 1989; Kollerand Smithies, 1992). Cultured cells or their nuclei may then betransferred into Drosophila (Okada et al., 1974; Van Deusen, 1977).

[0049] Previous attempts at culturing germline stem cells utilized the40 germline cells present in each embryo at a certain stage ofdevelopment. But no dpp was provided, and these cells differentiated inculture (Allis et al., 1979). Inducing BMP expression in cells of suchcultures or adding exogenous BMP to them would be a simple way ofmaintaining and/or propagating germline stem cells in vitro.

[0050] A BMP may also be used in replacement of, or combination with,known stem growth factors such as, for example, fibroblast growth factor(FGF), leukemia inhibitory factor (LIF), and steel factor (SF). Thus,BMP activity as observed herein might also be demonstrated using thetechniques taught in U.S. Pat. Nos. 5,453,357 and 5,690,926.

[0051] Ex vivo culturing of stem cells with stimulation of BMP signalingonly performed outside the body is preferred to avoid systemic effectsof BMP signaling on the organism.

[0052] Vascular or organ engineering may be accomplished with stem cellsthat differentiate into endothelium or parenchyma, respectively, with orwithout an implantable support (e.g., stent, hollow fiber or particle)on which stem cells have been coated or impregnated. If not autologouslytransplanted and in an organism with an immune system recognizinghistoincompatibility, transplantation of allogeneic or xenogeneic tissuemay require immunosuppression of the host (e.g., cyclosporine A or FK506treatment). Differentiation of stem cells into tissue with the activityand/or structure of adrenal gland, bone marrow, brain, liver, ovary ortestis, pancreas, peripheral neurons or glia, red or white blood cells,skeletal or smooth muscle, skin, thyroid gland, or combinations thereofis preferred.

[0053] One or more genes of the stem cell may be activated or inhibitedby chemical or environmental induction, antisense, ribozyme, chimericrepair vector, RNAi, or random/sequence-specific insertion. Ectopicexpression of a gene may be controlled in a particular spatial ortemporal manner, mimic pathologic or disease states, or createphenocopies of mutations in the endogenous gene. Homologousrecombination is preferred to achieve gene knockout or replacement (see,e.g., U.S. Pat. Nos. 5,569,824, 5,602,307, 5,614,396, 5,683,906, and5,830,682). For example, stem cells may be transfected with apolynucleotide, the polynucleotide or a portion thereof integrates intothe genome of transfected stem cells at a random site or in asequence-specific manner, homologous recombinants at a genetic loci ofinterest are selected, and the selected stem cells are transplanted intoa host organism. Physical introduction of polynucleotides (e.g.,biolistics, electroporation, microinjection) is preferred.Alternatively, insertion of P elements may be genetically engineered invivo or in vitro in a stem cell maintained and/or propagated accordingto the present invention to disrupt genes (cf. Zhang and Spradling,1994; Spradling et al., 1995).

[0054] TGF-β signaling has been shown to limit the growth of germlinecysts during Drosophila spermatogenesis (Matunis et al., 1997). Whenpunt or shn function is removed in clones of somatic cells that surroundgerm cells, cysts continue dividing after four rounds of mitosis(Matunis et al., 1997). However, these investigators did not addresswhether this pathway functions in male germline stem cells. In theembryo and imaginal discs, punt and shn can function downstream of dpp(Ruberte et al., 1995; Letsou et al., 1995; Arora et al., 1995; Griederet al., 1995), but it was not known whether dpp or another TGF-β familymember is utilized to send the signal. Clonal analysis of mutants in dppdownstream components in male germline stem cells, like those reportedhere in the ovary, could show whether Dpp and/or other TGF-β-likemolecules are required for their division and maintenance in the testis.

[0055] In mouse, the BMP family members BMP-2 and -4 are most closelyrelated to dpp, with greater than 75% identity, and can function torescue dpp mutants in embryos (Padget et al., 1993). Recently, bothgenes have been inactivated by homologous recombination, but thehomozygous embryos die too early to assess possible functions in thegonads (Winnier et al., 1995; Zhang and Bradley, 1996; reviewed byHogan, 1996b). Consistent with our findings, Lawson et al. (1999) reportthat BMP-4 affects the number of primordial germ cells; moreover, BMP4was needed in somatic tissue, and presumably stimulated BMP signaltransduction in germline cells, although this was not shown directly.The roles during spermatogenesis of two other BMP family members, BMP-8Aand BMP-8B, have been tested (Zhao et al., 1996; 1998). BMP-8B isrequired for the resumption of male germline cell proliferation in earlypuberty, and for germline cell survival in the adult, whereas BMP-8Aplays a role in the maintenance of adult spermatogenesis.

[0056] The “niche” hypothesis postulates that stem cells reside inoptimal microenvironments or “niches” (Schofield, 1978). When a stemcell divides, only one daughter can remain in the niche while the otherbecomes committed to differentiate. A stem cell within the niche wouldhave a high probability of self-renewal, but a low probability of entryinto the differentiation pathway. This model is consistent with theobservations that stem cells require the addition of growth factors forproliferation and differentiation in many in vitro culture systems(Potter and Loeffler, 1990; Morrison et al., 1997). The molecular natureof the microenvironment within a niche has yet to be defined in anysystem, although the Drosophila germarium appears to contain such aniche. Anteriorly, the stem cells abut terminal filament and cap cells,which both express hh, while only the latter express armadillo (arm) andwg (Forbes et al., 1996a; 1996b). Stem cell daughters lie more to theposterior, and probably directly contact inner germarial sheath cells,which express patched (ptc) and hh (Forbes et al., 1996b). Thisasymmetry in structure and signals may allow germline stem cells toreceive different levels of signals from their daughters. Consistentwith the existence of a niche, two wildtype stem cells in germaria thatrecently lost a marked mutant stem cell were occasionally observed,suggesting that a vacated niche could be reoccupied.

[0057] The existence of the germline stem cell niche is also consistentwith stem cell proliferation when local dpp is overexpressed. Underthese conditions, the size of the niche may be substantially enlarged.Conversely, reduction of dpp function may weaken the ability of theniche to maintain germline stem cells, leading to accelerated losses.These results suggest that dpp is an essential niche signal. However,dpp likely interacts with other signals from surrounding somatic cellsto make a functional niche for germline stem cells. Nonetheless, theidentification of dpp as a key niche signal should greatly facilitateefforts to culture Drosophila germline stem cells in vitro.

[0058] Technical limitations have previously prevented identification ofthe source of the dpp signal that is received by germline stem cells.Ideally, analysis of clones of a null dpp allele would reveal whichcells produce the signal. However, the somatic cells adjacent to thestem cells cease division early in ovary development and make inductionof specific small clones difficult. The pattern of dpp expression in thegermarium should also provide some insight into the origins of thesignal. However, the only available dpp-lacZ fusion line and whole mountin situ experiments failed to detect expression in the germarium,although follicle cell expression in late stage egg chambers wasobserved. We now show that somatic cells in the niche express dpp. Inmany systems, low levels of dpp expression are known to be sufficientfor biological effects so it may be sufficient to provide only lowlevels of BMP in the present invention.

[0059] In the Drosophila leg, antenna and genital discs, dpp and wg areinduced in the anterior compartment by hh, and the mutual repression ofdpp and wg restricts them to their appropriate domains (Brook and Cohen,1996; Jiang and Struhl, 1996; Chen and Baker, 1996). In vertebrate limbdevelopment, sonic hedgehog (shh) can induce the expression of BMP-2(Johnson and Tabin, 1995). The somatic terminal filament, cap, and innersheath cells express hh and lie adjacent to the germline stem cells(Forbes et al., 1996a, 1996b). wg and dpp expression may be induced byhh, and signal to germline stem cells for their proliferation andmaintenance. The data indicate that these and possibly additionalsignals from the anterior somatic cells define a niche for germline stemcells at the tip of germarium. Thus, agents which modify hedgehogsignaling may be used to alter local BMP signaling, thereby regulatingstem cell maintenance and/or propagation.

EXAMPLES Example 1 Ectopic Dpp Expression Induces Germ Cell Tumors

[0060] To assess whether Dpp can regulate germline stem cells in theDrosophila adult ovary, Dpp was ectopically expressed in the germariumusing hsp70-GAL4 (hs-GAL4) and UAS-dpp (Brand and Perrimon, 1993). Todistinguish different cell types in the germarium, we used anti-Hts andanti-Vase antibodies to visualize somatic and germline cells,respectively. The anti-Hts antibody also recognizes spectrosomes andfusomes in the germline cells of the germarium (see de Cuevas et al.,1997).

[0061] Only germline stem cells and cystoblasts have a big roundspectrosome, while cysts have a characteristic branched fusome. In thewildtype germarium, two germline stem cells are more anteriorly locatedthan cystoblasts. Developing cysts in germarial regions 1 and 2a (i.e.,the anterior half), which are more posterior than germline stem cellsand cystoblasts, are connected by fusomes. In the germarial regions 2band 3, both lens-shaped and round cysts span across the germarium, andbecome surrounded by somatic follicle cells. Fusome structures begindegeneration in these older cysts.

[0062] The germaria from hs-GAL4 females subjected to heat shock, andthose from females carrying hs-GAL4 and UAS-dpp in the absence of a heatshock, were indistinguishable from wildtype. In these heat shock-treatedgermaria, large single germline cells filling the corresponding wildtypegermarial regions 1 and 2a contained spectrosomes but showed no evidenceof cyst formation. In the corresponding wildtype regions 2b and 3, bothlens-shaped and round cysts were observed that probably derived fromdifferentiated cystoblasts or cysts that had formed before the initialheat shock.

[0063] Consistent with this interpretation, after 4-5 days of heatshock, all germ line cells in the corresponding regions 1 and 2 weresingle cells containing spectrosomes and developing cysts containingbranched fusomes were rarely detected. Only somatic follicle cells weredetected, there were no germline cells. This phenotype is very similarto that of bam and benign gonial cell neoplasm (bgcn) mutants (McKearinand Spradling, 1990; Gateff and Mechler, 1989).

[0064] Here, instead of the wildtype number of two or three germlinestem cells per ovariole, dozens were present in a single ovariole.Moreover, the number present was 2-3 times greater after 4-5 days thanafter 3 days. Because there are 16 ovarioles per ovary and two ovariesper female fly, all of the above numbers should be multiplied by 32 tocalculate the number of female germline stem cells per fly. The germlinestem cells proliferate following induction of dpp to form a large massof normal appearing, normal functioning germline stem cells. Theproliferating cells were shown to be germline stem cells based on (1)general size and appearance, (2) fusome morphology, (3) expression ofthe germ cell-specific gene vasa, (4) absence of expression ofcytoplasmic Bam (i.e., a sensitive indicator that germline stem cellshave differentiated into cystoblasts), and (5) ability to differentiatealong the normal pathway for germline cells following removal of dpp.

Example 2 Dpp-Induced Tumor Cells Resemble Germline Stem Cells

[0065] Cystoblasts and early mitotic cysts can be distinguished fromstem cells because the former express cytoplasmic Bam protein from thecystoblast stage to the end of the 8-cell stage cyst stage.Immunofluorescent staining of the wildtype germarium with anti-BamC andanti-α-spectrin antibodies document that cystoblasts and developingcysts, but not germline stem cells, express cytoplasmic Bam protein.Immunofluorescent staining of dpp-induced germaria with anti-BamC andanti-α-spectrin revealed that amplified single germline cells failed toexpress the cytoplasmic Bam protein. In dpp-induced germaria, a few,rare BamC-positive cells were observed that appeared to be growingcysts. These data show that the large number of single germline cellsinduced by dpp overexpression resemble stem cells rather thandifferentiated cystoblasts.

[0066] To determine that this absence of BamC-staining was not due togrowth arrest of the accumulated single germline cells, dpp-inducedgermaria were stained with anti-BrdU and anti-α-spectrin antibodiesfollowing incorporation of the nucleotide analog BrdU for one hour.Mitotically active germline cells in their S-phase of the cell cycle canincorporate BrdU. In the dpp-induced germaria, some single germlinecells incorporated BrdU, indicating that these single germline cellshave not undergone growth arrest.

[0067] These results show that the tumor cells induced by dppoverexpression continue to divide, and resemble stem cells in theirfusome morphology and absence of Bam protein. They represent anincreased number of germline stem cells.

[0068] To determine if these dpp-induced stem cells retain the capacityto differentiate, their behavior was examined. hs-GAL4/UAS-dppDrosophila were induced by four days of heat shock-treatment, and thenreturned to room temperature for 2 or 4 days prior to staining withanti-Hts and anti-Vase antibodies. Germline cysts were observed startingto form two days after the temperature down-shift and always formedinitially in the most posterior region of the tumor. Many 16-cell cystswere seen 4 days after the shift back to room temperature. Based ontheir location and number, these cysts must derive from dpp-inducedgermline stem cells, rather than from stem cell divisions that occurafter the downshift. But not all the dpp-induced germline stem cellswere able to form complete cysts, because some ovarioles contained cystswith one, two, four, or eight cells in region 3.

Example 3 Overexpressed dpp Acts Directly on Germline Stem Cells

[0069] Two different models could explain the Dpp effect on germlinestem cells: direct signaling to the germline stem cells and relaysignaling. The relay signaling model predicts that ectopic Dpp turns ona secondary signal in the somatic cells surrounding germline stem cells.

[0070] To directly test the relay model, the hs-GAL4/UAS system was usedto activate Dpp type I receptors. The hs-GAL4/UAS system can express atarget gene at high levels in somatic cells of the adult ovary, but notin germline cells (Manseau et al., 1997). Both activated tkv (tkv*)(Nellen et al., 1996) and activated sax (sax*) (Des et al., 1998) havebeen shown to mimic dpp signaling pathway activation in manydevelopmental processes.

[0071] Overexpression of activated Dpp type I receptors in the somaticcells of the germarium does not mimic the effect of ectopic dppexpression. Flies of the following genotypes were subjected to heatshock-treatment for three days, and germaria were subsequently labeledwith anti-Vase and anti-Hts antibodies: hsGAL41UAS-sax*,hsGAL4/UAS-tkv*, and UAS-sax*/+;hsGAL4/UAS-tkv*. Two independent linescontaining the UAS-tkv* and UAS-sax* insertions at different chromosomalsites were tested. When activated sax* or tkv*, or both, wereoverexpressed in the somatic cells of the germarium using hs-GAL4, thesame driver for dpp overexpression, no germline stem cell proliferationwas observed. But egg chamber budding was frequently affected in region3 cysts in the hsGAL4/UAS-tkv*, and UAS-sax*/+;hsGAL4/UAS-tkv* lines,suggesting that somatic follicle cell function was defective at a laterstage.

[0072] These results suggest that relay signaling, regardless of itsmechanism, is by itself not sufficient to inhibit germline stem celldifferentiation. Since overexpressed Dpp does not appear to act by arelay signal, it likely acts directly on germline cells via functionalDpp receptors to inhibit cystoblast differentiation.

Example 4 Dpp and Sax are Required for Germline Stem Cell Division andMaintenance

[0073] To directly test the role of dpp, we examined mutations thatreduce its function and that of the Dpp receptor sax. Dpp signaling isessential at many points during Drosophila development. Severaltemperature-sensitive allelic combinations of dpp mutants, includingdpp^(e90)/dpp^(hr56) and dpp^(hr4)/dpp^(hr56), can develop into adultsat 18° C. (Wharton et al., 1996). These heteroallelic combinationsallowed us to examine the mutant phenotypes of dpp in the germariumafter the shift to 28° C. Forty to 50% of germaria from these genotypesexamined one week after the temperature shift were significantly smallerthan heterozygotes, and more severe reductions were seen in olderfemales maintained at the higher temperature. To determine if stem cellswere being lost, ovaries from the mutant females were stained withanti-Hts and anti-Vase antibodies and the number of stem cells in eachovariole were directly counted (Table 1). There was a dramatic reductionin germline stem cell number in both tested genotypes over a two weekperiod. The stronger of the two, dpp^(hr4)/dpp^(hr56), almost completelyeliminated stem cells within two weeks. This combination produces manyfewer adult flies and is known to disrupt embryonic development moreseverely than dpp^(e90)/dpp^(hr56) (Wharton et al., 1996).

[0074] If the mutations act specifically on germline stem cells,cystoblasts and cysts should continue to divide and develop. To examinethis, the morphology of fusomes in the mutant ovarioles were analyzed.Ovarioles from the Dpp receptor mutant sax^(P), which has a weakereffect on stem cell number were also studied. The timing of stem cellloss is expected to vary among individual germaria, because stem cellloss is a random process (Margolis and Spradling, 1995).

[0075] Control germaria from one week-old sax^(P)/+ and dpp^(e90)/CyOP23females were double labeled with anti-Vase and anti-Hts antibodies. Inboth cases, germaria from one week-old females heterozygous for the dppor sax alleles generally contained two stem cells at the anterior. Themutant germania were also double labeled with anti-Vase and anti-Htsantibodies.

[0076] Mutant sax^(P)/sax^(P) germaria from one week-old females weresmaller than wildtype. In one case, two stem cells were observed but thenumber of cysts was reduced. In another case, one stem cell remained andregions 1 and 2a were much reduced as indicated by the start of region2b. This indicates that stem cells were being lost and their divisionslowed.

[0077] Many germaria in two week-old mutant sax^(P)/sax^(P) females hadlost both stem cells and no mitotic cysts were present, although cystsand egg chambers at later developmental stages remained (e.g., the mostanterior cyst corresponding to region 2b).

[0078] Mutant dPP^(e90)/dPP^(hr56) females showed a more rapid loss ofstem cells at 28° C. Such germaria frequently contained one or zero stemcells after one week. In one case, only one stem cell and no mitoticcysts were found; the most anterior cyst contained 16 cells. In anothercase, no stem cells were present; an 8-cell cyst and a 16-cell cyst layat the anterior. After two weeks, most ovarioles lacked stem cellsentirely, but some still contained 16-cell cysts or older follicles.

[0079] Because normal cystocyte development continued throughout thegermarium, the effects of these mutations appear to be limited largelyto stem cell division and maintenance. Some abnormalities in a laterprocess, egg chamber budding, were observed. Stem cell loss might becaused by either cell death or differentiation. Apoptotic cells were notobserved in the most anterior region of these germaria where germlinestem cells are located based on DAPI staining.

[0080] These results indicate that a reduction in the level of dppsignaling promotes the differentiation of germline stem cells intocysts, and thus causes stem cell loss. Consistent with previous studies(Twombly et al., 1996), we observed some partially ventralized eggs withanterior defects in these dpp mutants and the sax^(P) mutant.

Example 5 Put, Tkv, Mad, Med, and Dad are Required Cell-Autonomously forGermline Stem Cell Maintenance.

[0081] To demonstrate definitively that dpp signaling was received bythe germ line, studies were conducted to assess whether components ofthe signal transduction pathway are autonomously required in thesecells. Flp-induced mitotic recombination was employed to generate markedclones homozygous for loss-of-function mutations in the germline stemcells of adult ovaries (see Experimental Procedures). Genes downstreamof dpp in the signal transduction pathway are required in the germlinestem cells for their division and maintenance. Germaria lacking orbearing stem cell clones of the indicated genotypes were generated, andthen labeled with anti-lacZ and anti-Hts antibodies. Marked stem cellsand their progeny cysts were indicated by the absence of lacZ protein.

[0082] Clones were marked using armadillo-lacZ, which is stronglyexpressed in all cells within the germarium when wildtype flies are notsubjected to heat shock. Stem cell clones can be recognized because onlystem cells persist in the germarium more than 5 days after a mitoticrecombination event (Margolis and Spradling, 1995). As recombinationevents can take place only in mitotically active adult cells, thismethod will not produce mutant clones in the terminally-differentiatedterminal filament, cap cells, and inner sheath cells. Consequently, thisapproach excludes potential complications due to mutant clones in thesesurrounding somatic cells, allowing the autonomous function of genes tobe tested in germline stem cells. This method has three major additionaladvantages. Firstly, the persistent mutant clones can be studied over along period of time allowing germline stem cell maintenance to bequantified. Secondly, the existence of both a mutant and a wildtype stemcell side-by-side in the same germarium provides a control for theeffects of gene removal by direct comparison. Thus, the relativedivision rates of these two stem cells can be determined simply bycounting the number of mutant and wildtype cysts in germania with onemutant and one wildtype stem cell. Finally, germline stem cell-specificeffects of the mutations can be assessed by looking at the developmentalstatus of marked cystoblasts, cysts, and egg chambers.

[0083] Germline stem cell clones of punt-, tkv-, mad-, Med-, and Dad-were generated by subjecting females of the appropriate genotype to heatshock and examining their ovaries beginning one week later. Stem cellsin the Drosophila ovary have a finite life span with a half-life ofabout 4.6 weeks (Margolis and Spradling, 1995; Table 2). In contrast towildtype clones, stem cells mutant for each of the tested genes (exceptDad) were lost more rapidly (Table 2). For example, after one week, thepunt¹³⁵ mutant germline stem cell was either still present or had onlyrecently been lost, as indicated by the presence of relatively youngmutant cysts. However, after two weeks, the punt¹³⁵ mutant germline stemcell had usually been lost and only a few advanced mutant cystsremained.

[0084] mad¹² mutant stem cells were lost even more rapidly. After oneweek, the mad¹² mutant germline stem cell sometimes remained, but didnot proliferate well as indicated by the lack of progeny cysts. Morefrequently, the germline stem cell was already lost and a more developedcyst (or cysts) was observed. After two weeks, mad¹² mutant germlinestem cells rarely remained so there were no mutant cysts, but oldermutant egg chambers were present. Surprisingly, two wildtype germlinestem cells were occasionally observed after the mutant stem cell waslost. These results indicate that the dpp signal directly acts ongermline stem cells to regulate their maintenance. However, no effectswere observed on the formation of 16-cell cysts or the subsequentdevelopment of germline cells.

[0085] Unlike the other tested genes, Dad is a negative regulator of dppsignaling. The Dad gene is induced by the dpp signaling pathway andantagonizes the function of dpp (Tsuneizumi et al., 1997). The Dad²⁷¹⁻⁶⁸allele is a severe allele in which the entire C-terminal conserveddomain was deleted (Tsuneizumi et al., 1997). Strikingly, germline stemcells mutant for Dad²⁷¹⁻⁶⁸ were not lost (e.g., a mutant germline stemcell and its progeny cysts may be present), even if both germline stemcells lacked this gene (e.g., two mutant germline stem cells and anormal complement of progeny cysts were present). No turnover could bedetected even after three weeks of clone induction, suggesting thatincreasing dpp signaling can prolong germline stem cell lifetime.

[0086] To compare the magnitude of the effects of different mutations onstem cells, the half-life of mutant germline stem cells was measured(Table 2; Experimental Procedures). punt¹⁰⁴⁶⁰ is a hypomorphic allele ofthe Dpp type II receptor whereas punt¹³⁵ is a strong allele (Arora etal., 1995; Letsou et al., 1995). In punt¹⁰⁴⁶⁰ clones, germline stem cellhalf-life was reduced from about 4.6 to 0.90 weeks, whereas the strongerpunt¹³⁵ allele reduced germline stem cell half-life to about 0.41 weeks.tkv⁸ is a strong allele of the type I receptor (Brummel et al., 1994;Nellen et al., 1994; Penton et al., 1994). tkv⁸ stem cell clones reducedgermline stem cell half-life to about 0.69 weeks. Clones of two allelesof the downstream signal transducer, mad⁹ and mad¹², reduced germlinestem cell half-life to 2.5 weeks and 0.25 weeks, respectively.Consistent with this observation, mad is a much stronger allele thanmad⁹ (Sekelsky et al., 1995). Med²⁶ is a strong allele of anotherdown-stream transducer (Des et al., 1998). Med²⁶ germline stem cellsturned over with a half-life of about 0.38 weeks.

Example 6 Punt, Tkv, Mad, Med Are Required Cell-Autonomously toStimulate Germline Stem Cell Division.

[0087] To further define the role of the dpp pathway in the regulationof germline stem cell division, the number of mutant and wildtype cystsin germaria carrying one mutant and one wildtype germline stem cell werecompared. Since each cyst represents one germline stem cell division,counting the number of wildtype and mutant cysts allowed the measure ofrelative germline stem cell division rates. All germaria that stillretained a mutant germline stem cell from all three time points werecounted and compared to the number of wildtype cysts. In controlscontaining a marked but genetically wildtype germline stem cell,approximately 50% of cysts were marked, indicating that two germlinestem cells are present in one week-old adult germaria and divide atsimilar rates (see Table 2).

[0088] As expected based on previous experiments punt-, tkv-, mad-, andMed- mutant germline stem cells all divided more slowly than wildtype(see Table 2). While the relative division rate of marked wildtypegermline stem cells was about 0.93, the rates in the tested genotypesranged from about 0.21 to 0.60. These reductions mostly correlated withthe known strength of these alleles, and with their effects on germlinestem cell maintenance. However, both punt¹⁰⁴⁶⁰ and punt¹³⁵ mutantgermline stem cells proliferated about three-fold slower than thewildtype, despite the fact that they differ in strength. Differencesbetween the effects of these mutants on maintenance and division mayreflect branch points in the pathway, and may suggest that at least oneadditional type II receptor also mediates germline stem cell behavior.Interestingly, Dad²⁷¹⁻⁶⁸ mutant germline stem cells, which were morestable than wildtype, divided at a similar or slightly slower rate thanwildtype ones. These results demonstrate that components of the dppsignaling pathway are required autonomously for the proliferation ofgermline stem cells.

[0089] As shown previously, cysts produced in the presence ofoverexpressed dpp driven by hs-GAL4 always contained 16 cells. To verifythat dpp signaling is not involved in regulating the cystoblast andcystocyte divisions, the number of germline cells in individual cystsmutant for punt¹⁰⁴⁶⁰, mad⁹, mad¹², Med²⁶, and Dad²⁷¹⁻⁶⁸ were counted. Inevery case, these cysts contained 16 cells, including a single oocyte.Therefore, the dpp signaling pathway specifically acts on stem cellswithin the germ line.

Example 7 dpp is Expressed in Differentiated Somatic Cells SurroundingGermline Stem Cells.

[0090] To directly localize the source of the Dpp signal, a whole mountmRNA in situ hybridization was performed to visualize expression of thedpp gene on two day-old wildtype females which were dissected and fixed.A standard protocol was used (Yue and Spradling, 1992) except thatprotease digestion was performed at 50 gm/ml for 5 min. No staining wasobserved using the dpp sense RNA probe as a control. Under the sameconditions, the dpp anti-sense probe detected the dpp mRNA in the innersheath cells and cap cells adjacent to germline stem cells, and in theposterior somatic follicle cells, but not in germline and terminalfilament cells. These expression data further support our finding thatsurrounding differentiated somatic cells constitute a niche for germlinestem cells.

Example 8 A Lost Germline Stem Cell can be Replaced by the Daughter ofthe Other Stem Cell in the Same Germarium.

[0091] To provide further evidence that the stem cells in the ovariolereside within a niche, we showed that lost germline stem cells can bereplaced and function as germline stem cells by cells that wouldotherwise differentiate. In one week-old germaria in which one stem cellis marked, the marked cell contributes almost 50% of cysts, suggestingthere are an average of two germline stem cells per germarium. Since wehave shown that wildtype germline stem cells turn over with a half-lifeof 4.6 weeks, ovarioles containing only one or zero germline stem cellswould arise at a predictable rate unless they are replaced. For example,after 4.6 weeks, 25%, 50% and 25% of the germaria are expected to havetwo, one, and zero germline stem cells. In contrast, we observed thatmore than 71% of five week-old germaria still contain two germline stemcells, 20% contained one germline stem cell, and 9% contained none.These results were unexpected and demonstrate that following loss,germline stem cells are replaced 62% of the time over this time period.

[0092] To determine how replacement occurs, we identified ovarioleswhere a marked stem cell had just been lost and was in the process ofbeing replaced Such ovarioles contain a marked cystoblast and markeddeveloping cysts, but no marked stem cell. We observed an unusualdivision of the remaining stem cell in a plane perpendicular to the axisof the ovariole. Such a division would place the stem cell daughter inthe same location as the recently lost stem cell. Normally, a germlinestem cell divides along the anterior-posterior axis, and the posteriordaughter differentiates into a cystoblast. These findings indicate thatthe fate of the stem cell daughter is determined by the environment.This environment within the niche maintains the stem cell fate, whilethe environment more posteriorly in the ovariole promotesdifferentiation as a cystoblast.

Example 9 Dpp is Required for Maintenance of Male Germline Stem Cells

[0093] Temperature sensitive dpp mutant genotypes were generated bycrossing dpp^(hr56)/CyO with dpp^(hr4) and dpp^(e90)/CyO.Temperature-sensitive punt mutant males were generated by crossingpunt¹⁰⁴⁶⁰/TM3 with punt¹³⁵/TM3 Sb. The dPP^(hr56)/dPP^(e90),dpp^(hr56)/dpp^(hr4) and punt¹⁰⁴⁶⁰¹/punt¹³⁵ adult males were raised at28° C. (i.e., the restrictive temperature) for one week. Heterozygotescontrols were also examined at 28° C.; testes were dissected out andstained with rabbit anti-Vasa and mouse anti-Hts antibodies.Cy3-conjungated goat anti-rabbit and FITC-conjungated goat anti-mousesecondary antibodies were used to visualize the Vasa protein (red) andHts protein (green) with a Leica TCS-NT confocal microscope. Thegermline stem cells are located at the tip of testes, and can berecognized by their expression of Vasa protein (red) and also containround fusomes (yellow), and by their association with hub cells (green).The differentiated germline cells lie more distant from the tip, andalso contain either round fusomes or branched fusomes.

[0094] To directly show that dpp regulates male germline stem cells inDrosophila testes, the number of stem cells was examined indpp^(hr56)/dpp^(e90), dpp^(hr56)/dpp^(hr4) and punt¹⁰⁴⁶⁰/punt¹³⁵ mutanttestes under restrictive conditions, and compared to heterozygotecontrol testes. In a heterozygous testis (control), there were betweenseven and nine germline stem cells located adjacent to somatic hub cellsand contain round fusomes like their counterparts in the ovary; thesetestes were full of developing germline cysts and primary spermatids.After one week at the restrictive temperature, there were still overseven germline stem cells. These values are indistinguishable from thetypical wildtype testis. In contrast, one week-old dpp^(hr56)/dpp^(e90),dpp^(hr56)/dpp^(hr4) and punt¹⁰⁴⁶⁰/punt¹³⁵ mutant testes from males thathad been raised at the restrictive temperature contained a reducednumber of germline stem cells, ranging in number from 2-7 per testis. Asa consequence of this loss, these testes were also significantly smallerthan the controls and contained fewer developing germline cysts andprimary spermatids. Because a testis starts with a much larger number ofgermline stem cells than an ovariole, complete loss would not beexpected within one week even if they require dpp and punt to the samedegree as female germline stem cells. These results demonstrate that dppand punt are required for maintaining male germline stem cells.

Example 10 Shn is Required for Germline Stem Cell Maintenance

[0095] schnurri (shn) encodes a zinc-finger protein homologous to humanMPB1. It is required for dpp signaling in the Drosophila embryo (Aroraet al., 1995; Greider et al., 1995). Mutant shn germline stem cellclones were generated as described above. FRT42D arm-lacZ/FRT42Darm-lacZ virgin females were crossed to FRT42D shn/CyO andFRT421D+/FRT42D+ (control) males, respectively. Two day-old adultnon-CyO females carrying an arm-lacZ transgene in trans to the shnmutant-bearing chromosomes were heat shocked twice at 37° C. for 60 mineach separated by eight hours. Germline stem cells were examined in thesame manner as described above. These results demonstrate that shn isalso required in germline stem cells for their maintenance and division.

Experimental Procedures

[0096] A description of materials and methods useful for practicing thepresent invention is given in the following general references: Lindsleyand Grell (Genetic Variations of Drosophila melanogaster, Carnegie Inst.of Wash., 1968); Ashburner (Drosophila: A laboratory handbook and Alaboratory manual, Cold Spring Harbor Lab., 1989); Lindsley and Zimm(The Genome of Drosophila melanogaser, Academic, 1992); Bate and Arias(The Development of Drosophila melanogaster, Cold Spring Harbor Lab.,1993); and Greenspan (Fly Pushing: The Theory and Practice of DrosophilaGenetics, Cold Spring Harbor Lab., 1997). Drosophila stocks may beobtained from the Bloomington Stock Center at Indiana University.Information relevant to Drosophila genetics and molecular biology,including recombinant clones and nucleotide/amino acid sequencesobtained through the Drosophila genome project, is publicly available inthe FLYBASE relational database (see Nucl. Acids Res. 27, 85-88, 1999).

[0097] Drosophila Stocks and Genetics

[0098] The following fly stocks used in this study were described eitherin the FlyBase or otherwise specified: tkv⁸; punt¹⁰⁴⁶⁰ and punt¹³⁵; mad⁹and mad¹²; Med²⁶ (Des et al., 1998); Dad²⁷¹⁻⁶⁸; sax^(P); dpp^(hr56)dpp^(hr4), dpp^(e90); UAS-dpp; hs-GAL4; HSFlp; FRT40A armadillo-lacZ andHSFLP;FRT82B armadillo-lacZ (Lecuit and Cohen, 1997); UAS-tkv*(activated) and UAS-sax* (activated) on both chromosomes 2 and 3 (Des etal., 1998). Most stocks were cultured at room temperature. To maximizetheir effects, sax^(P) and dpp mutants were cultured at 28° C. for 1-2weeks.

[0099] Generating Mutant Germline Stem Cell Clones and Overexpression

[0100] Clones of mutant cells were generated by Flp-mediated mitoticrecombination as described previously (Xu and Rubin, 1993). To generatethe stocks for stem cell clonal analysis, +FRT40A/CyO, tkv⁸ FRT40A/CyO,mad⁹ FRT40A/CyO, and mad¹² FRT40A/CyO males were mated with virginfemales w HSFlp1; armadillo-lacZ FRT40A, respectively. FRT82B Med²⁶/TM3Sb, FRT82B punt¹³⁵/TM3 Sb, FRT82B punt¹⁰⁴⁶⁰/TM3 Sb, FRT82B Dad²⁷¹⁻⁶⁸/TM3Sb males were mated with virgin females w HSFlp1; FRT82B armadillo-lacZ,respectively. Two day-old adult non-CyO or non-Sb females carrying anarmadillo-lacZ transgene in trans to the mutant-bearing chromosome wereheat shocked at 37° C. for 60 min. The females were transferred to freshfood every day at room temperature, and ovaries were removed one week,two weeks, or three weeks after the last heat shock-treatment andprocessed for antibody staining.

[0101] To construct the stocks for overexpressing dpp and activatedreceptors, the hsGAL4 virgins were crossed with UAS-dpp, UAS-tkv*/CyO,UAS-tkv*/TM3 Sb, UAS-sax*/CyO, UAS-sax*/TM3 Sb, UAS-tkv*/CyO;UAS-sax*/TM6, UAS-sax*/CyO; UAS-tkv*/TM6 males, respectively. Thefemales which did not carry balancer chromosomes were heat shocked at37° C. for 30 min each time with the interval of 12 hr for 3-5 days.

[0102] Calculations

[0103] To determine stem cell life spans, stem cells were marked in oneto two day-old females of the appropriate genotype by a single heatpulse. Subsequently, ovaries were dissected from some of the femalesone, two, and three weeks later and stained with anti-Hts and anti-lacZantibodies. The percentage of germaria containing a marked stem cell wasdetermined by counts of 60-200 germaria at each time point, and used tocalculate the stem cell half-life.

[0104] To measure stem cell division rates, we determined the relativenumber of wildtype and mutant cysts in germaria that contained onewildtype and one mutant stem cell. A relative division rate of 1.0 wouldindicate normal division. For a given genotype, these values weresimilar at each time point, and the average is presented in Table 2.Marked wildtype stem cells gave a value of 0.93 rather than 1.0 probablydue to a small fraction of germaria that contained three rather than twogermline stem cells.

[0105] To measure stem cell loss, germaria with two, one, or no germlinestem cells, were counted from the ovaries of the one and two week-oldfemales. Heterozygous females carrying one copy of the mutant gene incombination with a CyO balancer chromosome containing a dpp transgene(Hursh et al., 1993) served as a control. Values are expressed as thepercentage of ovarioles with the indicated stem cell compositions.

[0106] Immunohistochemistry

[0107] The following antisera at the indicated dilutions were used:polyclonal anti-Vasa antibody (1:2000) (Liang et al., 1990); monoclonalanti-Hts antibody IB 1 (1:5) (Zacci and Lipshitz, 1996); poLyclonalanti-α-spectrin antibody (1:100) (Byers et al., 1987); rat anti-Bamantibody (1:100) (McKearin and Ohlstein, 1995); monoclonal anti-BrdUantibody (1:50) (Becton-Dickinson); polyclonal anti-β-galactosidaseantibody (1:1000) (Cappel). Labeling with BrdU was carried out for 1hour at room temperature as described by de Cuevas and Spradling (1998).All photomicrographs were taken using a Leica TCS-NT confocalmicroscope. TABLE 1 Dpp is Required for Germline Stem Cell Maintenance.One week Two weeks No One Two No One Two Genotypes GSC GSC GSC GSC GSCGSC dpp^(e90)/CyOP23  0.0%  4.4% 95.6%  0.5% 17.5% 82.0%  (0)  (5) (108) (1)  (36) (168) dpp^(hr4)/CyOP23  0.0%  3.5% 96.5%  1.5% 23.9% 74.6% (0)  (6) (165)  (3)  (48) (150) dpp^(e90)/dpp^(hr56) 16.0% 29.3% 54.7%47.3% 39.8% 12.9% (17) (31)  (58) (140) (118)  (38) dpp^(hr4)/dpp^(hr56)18.1% 33.9% 48.0% 98.4% 1.6%  0.0% (22) (41)  (58) (122)  (2)  (0)

[0108] The percentage of ovarioles with zero, one or two germline stemcells is given for each genotype. Actual numbers are given inparentheses. ^(a)P23 is a dpp transgene on the CyO chromosome (Hursh etal., 1993). TABLE 2 Downstream Components of the dpp Pathway areRequired in Germline Stem Cells for their Maintenance and Division.Percent of Germaria^(a) GSC^(b) Relative^(c) with a Marked GSC Half-LifeDivision Strains 1 week 2 weeks 3 weeks (weeks) Rate Control 37.7 (138)34.4 (161) 27.5 (160) 4.6 0.93 (1410) punt¹⁰⁴⁶⁰ 43.2 (118) 26.4 (182) 9.5 (116) 0.90 0.36 (1126) punt¹³⁵ 27.4 (95)  5.1 (138)   0 (114) 0.410.37 (329) tkv⁸ 38.6 (132) 16.4 (176)  6.1 (197) 0.69 0.29 (744) mad⁹43.6 (g4) 29.3 (208) 25.8 (155) 2.5 0.60 (1116) mad¹² 17.8 (124)   0(108)  0.7 (136) 0.25 0.21 (214) Med²⁶ 23.8 (172)  7.3 (110)   0 (122)0.38 0.39 (512) Dad²⁷¹⁻⁶⁸ 28.0 (107) 32.6 (86) 32.3 (62) >>4.6 0.84(770) Control 41.3 (235) 33.8 (185) 32.1 (379) 4.7 1.16 (316) shnP 38.9(126) 23.7 (228) 16.5 (332) 2.2 0.53 (331)

[0109] While the present invention has been described by what ispresently considered to be practical and preferred embodiments, it is tobe understood that variations in the claimed invention will be obviousto skilled artisans without departing from the novel aspects of thepresent invention and that such variations are intended to come withinthe scope of the claims.

[0110] For example, components of the dpp signaling pathway areconserved in structure (e.g., amino acid residues are identical orchemically analogous in a high proportion of positions when sequencesare aligned) and function such that mammalian proteins can rescueDrosophila mutant phenotypes which result from mutations in homologousgene of the pathway. Equivalents to the Drosophila genes and proteinsidentified herein, as well as mutants thereof, would be known to skilledartisans practicing the present invention by their similarity in aminoacid sequence (e.g., members of the TGF-β family) and/or their abilityto at least partially rescue mutant phenotypes or to create phenocopiesof such phenotypes.

[0111] Thus, the extent of legal protection will be determined by thelimitations recited in the allowed claims and their equivalents. Unlessexplicitly recited, other aspects of the present invention as describedin this specification do not limit the scope of the claims. In thisregard, the mechanisms of action suggested in the specification (e.g.,models for BMP signaling) are merely possible explanations for ourobservations while operation of the claimed invention is not necessarilydependent thereon.

[0112] All references, patent applications, and patents cited in thisdisclosure are hereby incorporated herein by reference in their entiretyand indicate the high skill of artisans in this field. In particular,some of the results shown above were published by Xie and Spradling inCell 94, 251-260 (1998) after the filing date of priority U.S. Appln.No. 60/094,008.

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We claim:
 1. A method for maintaining germline stem cells of Drosophilacomprising: (a) providing a population comprised of said germline stemcells, and (b) stimulating signal transduction by a bone morphogeneticprotein (BMP) signaling pathway in at least one cell of said population;wherein said stimulation maintains more germline stem cells in saidpopulation as compared to a population which has not had signaltransduction of said BMP signaling pathway stimulated.
 2. A methodaccording to claim 1, wherein said population is maintained in vivo andsaid Drosophila has been genetically engineered to stimulate said signaltransduction.
 3. A method according to claim 1, wherein said populationin maintained in vitro.
 4. A method according to claim 1, wherein saidgermline stem cells are from ovary.
 5. A method according to claim 1,wherein said germline stem cells are from testis.
 6. A method accordingto claim 1 further comprising obtaining said germline stem cells from anembryo.
 7. A method according to claim 1, wherein said BMP signalingpathway is stimulated by providing at least 10% more Decapentaplegic(Dpp) activity to said population than is present in wildtype.
 8. Amethod according to claim 1, wherein said BMP signaling pathway isstimulated by at least mutating a dpp gene to a gain-of-functionphenotype.
 9. A method according to claim 1, wherein said BMP signalingpathway is stimulated by at least providing a BMP to said population.10. A method according to claim 9, wherein said BMP is selected from thegroup consisting of Decapentaplegic (Dpp) protein, BMP-2, and BMP-4. 11.A method according to claim 1, wherein said BMP signaling pathway isstimulated by at least mutating a type I or type II decapentaplegic(dpp) receptor to a gain-of-function phenotype.
 12. A method accordingto claim 1, wherein said BMP signaling pathway is stimulated through atleast one serine/threonine kinase receptor which specifically recognizesa BMP.
 13. A method according to claim 12, wherein said BMP receptor isselected from the group consisting of Saxophone (Sax), Thick veins(Tkv), and Punt (Put).
 14. A method according to claim 1, wherein saidBMP signaling pathway is stimulated by altering activity of at least onesignal transducer for receptor binding to a BMP.
 15. A method accordingto claim 14, wherein said signal transducer is selected from the groupconsisting of Mothers against dpp (Mad), Medea (Med), Daughters againstdpp (Dad), Schnurri (Shn), and Brinker (Brk).
 16. A method according toclaim 1, wherein said BMP signaling pathway is stimulated by increasingexpression of BMP in a cell of said population.
 17. A method accordingto claim 16, wherein BMP expression is increased by hedgehog(hh)-activated transcription or wingless (wg)-activated transcription,and BMP signaling is increased in at least some of the germline stemcells.
 18. A method according to claim 1, wherein said population isfurther comprised of at least one somatic cell selected from the groupconsisting of terminal filament cell, cap cell, inner sheath cell, hubcell, and cyst progenitor cell.
 19. A method according to claim 1,wherein at least one germline stem cell is cultured in vitro in contactwith feeder cells expressing a bone morphogenetic protein (BMP).
 20. Amethod according to claim 1, wherein at least one germline stem cell iscultured in vitro in contact with at least some somatic niche cells. 21.A method according to claim 1, wherein signal transduction through saidBMP signaling pathway is stimulated by in vitro culturing said germlinestem cells with a feeder layer of somatic cells which stimulate BMPsignaling.
 22. A method according to claim 1, wherein signaltransduction through said BMP signaling pathway is stimulated by invitro culturing said germline stem cells in a culture medium whichstimulates BMP signaling.
 23. A method according to claim 1 furthercomprising maintaining at least one of said germline stem cells in apluripotent state.
 24. A method according to claim 1 further comprisingmaintaining at least one of said germline stem cells in a totipotentstate.
 25. A method according to claim 1 further comprising transferringat least one of said stimulated germline stem cells into a hostDrosophila.
 26. A method according to claim 25, wherein at least one ofsaid transferred germline stem cells contributes to two or moredifferentiated cell lineages of said host Drosophila.
 27. A methodaccording to claim 25, wherein at least one of said transferred germlinestem cells contributes to a germline cell lineage of said hostDrosophila.
 28. A method according to claim 1 further comprisingmutating at least one gene of said germline stem cell's genome.
 29. Amethod according to claim 1 further comprising introducing one or morepolynucleotides into said germline stem cell's genome.
 30. A methodaccording to claim 1 further comprising integrating a polynucleotide byhomologous recombination at a targeted genetic locus of said germlinestem cell.
 31. A method according to claim 1 further comprisingtargeting at least one gene of said germline stem cell for homologousrecombination, selecting at least one germline stem cell which hasundergone homologous recombination of said gene, and transferring saidhomologously recombinined germline stem cells into another Drosophilasuch that said targeted gene is genetically transmitted through saidanother Drosophila's germline.
 32. A cell population made by a methodaccording to claim 1, wherein there are at least ten germline stem cellsin said population for each germline stem cell present prior tostimulation of BMP signaling.
 33. A method for maintaining Drosophilastem cells comprising: (a) providing a population comprised of said stemcells, and (b) stimulating decapentaplegic (dpp) signaling such thatmore stem cells of said population are maintained as at least viable orundifferentiated as compared to a population of stem cells which has notbeen stimulated.
 34. A method of reducing or eliminating stem cells ortumor cells of an organism comprising: repressing signal transduction bya bone morphogenetic protein (BMP) receptor pathway such that said stemcells or tumor cells are reduced or eliminated.
 35. A method ofincreasing abundance of stem cells of an organism comprising:stimulating signal transduction by a bone morphogenetic protein (BMP)receptor pathway such that abundance of at least some stem cells isincreased.
 36. A method of increasing lifetime of stern cells of anorganism comprising: stimulating signal transduction by a bonemorphogenetic protein (BMP) receptor pathway such that said lifetime ofat least some stem cells is increased.